GPCRs also known as seven-transmembrane domain receptors comprise a large protein family of transmembrane receptors that sense molecules outside the cell and activate inside signal transduction pathways and ultimately cellular responses. GPCRs are involved in various physiological functions as well as many diseases, and are also the target of around half of all modem medicinal drugs. There are two principal signal transduction pathways involving the GPCRs: cAMP signal pathway and Phosphatidylinositol signal pathway.
The cytoplasmic tail region of GPCRs interacts to one of the three main classes of G proteins. G proteins are composed of a common βγ subunit and a specific α subunit. The role of the α subunit is well known for translating the extracellular cues to intracellular responses. G proteins are mainly classified into three categories based on the nature of their subunits. G proteins containing Gαs or Gαi subunits enhance or reduce the cAMP level respectively upon receptor stimulation through adenylate cyclase enzyme. In contrast, G proteins comprising Gαq subunit mobilize intracellular calcium ions upon activation of the receptor through a membrane bound phospholipase C enzyme. In recent years, Gβγ subunits turned out to be more than a silent partner. They too transmit the message through activation of ERK-MAPK pathways.
The molecular diversity of GPCR-mediated signal transduction pathway complicates the configuration of a common functional assay. The development of high through-put functional assays for GPCRs would greatly enhance the ability to discover and develop novel agonists and antagonists to this important superfamily of pharmaceutical targets. One approach for developing a high through-put functional GPCR assay is the use of reporter gene assays. Reporter gene constructs couple transcriptional enhancers that are regulated by various intracellular second messengers with appropriate promoter and reporter gene elements to produce a surrogate signal transduction system responsive to signaling pathways activated by various hormone receptors (Deschamps, Science, 1985 230:1174-7; Montminy, Proc. Nail. Acad Sci USA, 1986 83:6682-6686; Angel, Cell, 1987, 49:729-39; Fisch, Mol. Cell. Biol, 1989 9:1327-31). With the appropriate choice of transcriptional enhancers, promoters, and reporter genes, non-radiometric functional assays have been configured for Gαs coupled GPCRs (Konig, Mol. Cell. Neurosciences, 1991, 2:331-337; Chen, Anal. Biochemistry, 1995, 226: 349-354) and Gαq coupled GPCRs (Weyer, Receptor and Channels, 1993 1:193-200) that are amenable to high through-put screening technology.
Earlier, some assays have been developed for the identification of GPCRs. However, some disadvantages are associated with the traditional radioligand binding assay and FLIPR based functional assays. In traditional binding assay a) hazardous radioactive ligands are used; b) the mode of action of a molecule needs to be further investigated using a separate radioactive or cell based functional assay. In the FLIPR based functional assay (European patent application: EP1310800), fluorescent counts are measured within 5 seconds of compound injection which may not be sufficient to bring the compound in equilibrium with the receptor. As a result of shorter incubation of compounds in FLIPR assay, important compounds may be missed out or artefactual values may be generated.
In order to overcome the above mentioned disadvantages, we have developed novel functional assay for 5-HT2A, histamine H1 or adrenergic alpha 1b receptors based on measurement of intracellular cAMP levels by measuring reporter gene activity:                a) which eliminates the usage of hazardous radioactive ligands. Thus an environmental friendly approach can be implemented to identify novel compounds.        b) mode of action (agonist or antagonist) as well as binding affinity of the ligand (pKb or pEC50) are derived from the same experiment.        c) compounds are incubated for few hours to bring them in equilibrium with the receptor. Longer incubation period in reporter gene format is required to have a sustained activation of the receptor leading to the synthesis of cAMP and induction of the reporter gene.        
Thus, the reporter gene based assay may be more in line with the physiological conditions wherein the drug is allowed to interact with the target receptor in the body.